Many oncogenes are known to encode tyrosine-specific protein kinases presumably exerting their transforming effects by phosphorylating key substrates present in normal cells. Of the known tyrosine kinases and substrates, many are tightly linked to the plasma membrane-associated cytoskeleton. The proposed research is designed to identify new substrates of the transforming tyrosine kinase pp60v-src and to analyze the relevance of the phosphorylation to transformation. For this, cells expressing transforming wild type or non-transforming non-myristylated mutants of SRC will be examined. Proteins phosphorylated on tyrosine residues in transformed cells will be isolated by affinity chromatography using a monoclonal anti-phosphotyrosine antibody previously made in this laboratory. We show this antibody is far superior to others available. The proteins specifically eluted from this antibody column with the hapten phosphotyrosine will then be used as antigen to produce monoclonal antibodies to individual components. We have already produced antibodies to several new proteins in this way. We will also examine phosphotyrosine-containing peptides derived from transformed and non-transformed cells. Peptides will be sequenced and anti-peptide antibodies will be made to select phosphorylation sites. We will focus on those substrates for which there is a correlation with transformation. We have identified a 22K substrate which displays such a correlation. Antibodies to individual substrates will be used to determine the subcellular localization of the proteins by immunofluorescence microscopy and subcellular fractionation of normal and transformed chick embryo fibroblasts. We will isolate the 22K substrate from normal chick tissues and examine the association with other cytoskeletal proteins. Peptides from the 22K protein will be sequenced and used to search the protein sequence database for possible homology to other known proteins. These studies will advance our knowledge of both the membrane skeleton of fibroblasts and alterations in the cytoskeleton occurring upon malignant transformation.